Nitric oxide synthase inhibitors

ABSTRACT

The present invention relates to novel amidino compounds of formula (I),or a salt, solvate, or physiologically functional derivative thereof;wherein R1 is selected from C1-4 alkyl, C3-4 cycloalkyl, C1-4 hydroxyalkyl, and C1-4 haloalkyl; to a process for their manufacture, to pharmaceutical compositions containing them, and to their use in therapy, in particular their use as selective inhibitors of inducible nitric oxide synthase.

This Application is filed pursuant to 35 U.S.C. §371 as a United StatesNational Phase Application of International Application No.PCT/EP99/03583, filed May 27, 1999, which claims priority to GreatBritain Priority Patent Application Serial No. 9811599.1, filed May 30,1998.

The present invention relates to novel amidino compounds, to a processfor their manufacture, to pharmaceutical compositions containing them,and to their use in therapy, in particular their use as selectiveinhibitors of inducible nitric oxide synthase.

Nitric oxide is the endogenous stimulator of the soluble guanylatecyclase enzyme and is involved in a number of biological actions. Excessnitric oxide production is also thought to be involved in a number ofconditions, including septic shock and many inflammatory diseases. Thebiochemical synthesis of nitric oxide from L-arginine is catalysed bythe enzyme NO synthase. Many inhibitors of NO synthase have beendescribed and proposed for therapeutic use.

More recently, it has been an object in this field to provide NOsynthase inhibitors displaying selectivity for inducible NO synthase(iNOS) over endothelial NO synthase (eNOS) and/or neuronal NO synthase(nNOS).

Thus W093/13055 describes selective NO synthase inhibitors of formula

and salts, and pharmaceutically acceptable esters and amides thereof, inwhich:

R₁ is a C₁₋₆ straight or branched chain alkyl group, a C₂₋₆alkenylgroup, a C₂₋₆alkynyl group, a C₃₋₆cycloalkyl group or a C₃₋₆cycloalkylC₁₋₆alkyl group;

Q is an alkylene, alkenylene or alkynylene group having 3 to 6 carbonatoms and which may optionally be substituted by one or more C₁₋₃alkylgroups;

a group of formula —(CH₂)_(p)X(CH₂)_(q)— where p is 2 or 3, q is 1 or 2and X is S(O)_(x) where x is 0, 1 or 2, 0 or NR² where R² is H orC₁₋₆alkyl; or

a group of formula —(CH₂)_(r)A(CH₂)_(s)— where r is 0, 1 or 2, s is 0, 1or 2 and A is a 3 to 6 membered carbocylic or heterocyclic ring whichmay optionally be substituted by one or more suitable substituents suchas C₁₋₆alkyl, C₁₋₆alkoxy, hydroxy, halo, nitro, cyano,trifluoroC₁₋₆alkyl, amino, C₁₋₆alkylamino or diC₁₋₆alkylamino.

The co-pending International application WO 98/30537 also describescertain amidino compounds which are selective inhibitors of induciblenitric oxide synthase.

We have now found a novel class of compounds which as well as beingselective iNOS inhibitors, display advantages including that they have arelatively long half-life, are orally bioavailable when administered invivo, and may be prepared from relatively cheap starting materials.

Therefore, according to the present invention there is provided acompound of formula (I)

or a salt, solvate, or physiologically functional derivative thereof;

wherein R¹ is selected from C₁₋₄ alkyl, C₃₋₄ cycloalkyl, C₁₋₄hydroxyalkyl, and C₁₋₄ haloalkyl.

In formula (I), R¹ is preferably C₁₋₄ alkyl, most preferably, methyl.

The compounds of formula (I) include two chiral centres i.e. the carbonwhich bears the R¹ substituent and the asymmetric centre in the aminoacid group. It is intended that formula (I) includes all optical isomerseither in substantially pure form or admixed in any proportions. In apreferred aspect, the amino acid group is in the natural Lconfiguration. In a further preferred aspect the carbon bearing thegroup R¹ is in the R configuration. In the most preferred aspect, theamino acid is in the natural L configuration and the carbon bearing thegroup R¹ is in the R configuration. Throughout this specification, wherethe stereochemistry of two chiral centres within a molecule are given,the first configuration refers to the amino acid α carbon and the secondrefers to the carbon bearing the R¹ substituent, for example,stereochemistry designated (R,S) means (R)-stereochemistry at the aminoacid α carbon, and (S)-stereochemistry at the carbon bearing the R¹substituent.

Thus, in a further aspect, the present invention provides a compoundselected from:

S-[(R)-2-(1-iminoethylamino)propyl]-L-cysteine;

S-[(S)-2-(1-iminoethylamino)propyl]-L-cysteine;

S-[(R/S)-2-(1-iminoethylamino)propyl]-L-cysteine;

S-[(R)-2-(1-iminoethylamino)propyl]-D-cysteine;

S-[(S)-2-(1-iminoethylamino)propyl]-D-cysteine;

S-[(R/S)-2-(1-iminoethylamino)propyl]-D-cysteine;

S-[(R/S)-2-(1-iminoethylamino)butyl]-L-cysteine;

S-[(R/S)-2-(1-iminoethylamino,2-cyclopropyl)ethyl]-L-cysteine; and

S-[(R/S)-2-(1-iminoethylamino,3-hydroxy)propyl]-L-cysteine.

and salts, solvates, and physiologically functional derivatives thereof.

In a preferred aspect, the present invention providesS-[(R)-2-(1-iminoethylamino)propyl]-L-cysteine or a salt, solvate, orphysiologically functional derivative thereof. In a particularlypreferred aspect, the present invention providesS-[(R)-2-(1-iminoethylamino)propyl]-L-cysteine or a salt thereof.

It is to be understood that the present invention covers allcombinations of particular and preferred groups described herein.

Salts and solvates of compounds of formula (I) which are suitable foruse in medicine are those wherein the counterion or associated solventis pharmaceutically acceptable. However, salts and solvates havingnon-pharmaceutically acceptable counterions or associated solvents arewithin the scope of the present invention, for example, for use asintermediates in the preparation of other compounds of formula (I) andtheir pharmaceutically acceptable salts, solvates, and physiologicallyfunctional derivatives.

By the term “physiologically functional derivative” is meant a chemicalderivative of a compound of formula (I) having the same physiologicalfunction as the free compound of formula (I), for example, by beingconvertible in the body thereto. According to the present invention,examples of physiologically functional derivatives include esters,amides, and carbamates; preferably esters and amides.

Suitable salts according to the invention include those formed with bothorganic and inorganic acids or bases. Pharmaceutically acceptable acidaddition salts include those formed from hydrochloric, hydrobromic,sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic,trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic,methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic,and isethionic acids. Pharmaceutically acceptable base salts includeammonium salts, alkali metal salts such as those of sodium andpotassium, alkaline earth metal salts such as those of calcium andmagnesium and salts with organic bases such as dicyclohexyl amine andN-methyl-D-glucamine.

Pharmaceutically acceptable esters and amides of the compounds offormula (I) may have the acid group converted to a C₁₋₆alkyl, aryl, arylC₁₋₆ alkyl, or amino acid ester or amide. Pharmaceutically acceptableamides and carbamates of the compounds of formula (I) may have an aminogroup converted to a C₁₋₆alkyl, aryl, aryl C₁₋₆ alkyl, or amino acidamide or carbamate.

As mentioned above, the compounds of formula (I) are inhibitors of NOsynthase as demonstrated in the NOS inhibition assays below.

Therefore, compounds of formula (I) and their pharmaceuticallyacceptable salts, solvates, and physiologically functional derivativeshave use in the prophylaxis and treatment of clinical conditions forwhich an inhibitor of NO synthase is indicated, in particular, aninhibitor of iNOS. Such conditions include inflammatory conditions,shock states, immune disorders, and disorders of gastrointestinalmotility. The compounds of formula (I) and pharmaceutically acceptablesalts, solvates, and physiologically functional derivatives thereof mayalso be of use in the prophylaxis and treatment of diseases of thecentral nervous system including migraine.

By shock states is meant those resulting from overproduction of NO, suchas septic shock, haemorrhagic shock, traumatic shock, or shock caused byfulminant hepatic failure or by therapy with cytokines such as TNF, IL-1and IL-2 or therapy with cytokine-inducing agents, for example5,6-dimethylxanthenone acetic acid.

Examples of inflammatory conditions and immune disorders include thoseof the joint, particularly arthritis (e.g. rheumatoid arthritis,osteoarthritis, prosthetic joint failure), or the gastrointestinal tract(e.g. ulcerative colitis, Crohn's disease, and other inflammatory boweldiseases, gastritis and mucosal inflammation resulting from infection,the enteropathy provoked by non-steroidal antiinflammatory drugs), ofthe lung (e.g. adult respiratory distress syndrome, asthma, cysticfibrosis, or chronic obstructive pulmonary disease), of the heart (e.g.myocarditis), of nervous tissue (e.g. multiple sclerosis), of thepancreas (e.g. diabetes melitus and complications thereof), of thekidney (e.g. glomerulonephritis), of the skin (e.g. dermatitis,psoriasis, eczema, urticaria), of the eye (e.g. glaucoma) as well as oftransplanted organs (e.g. rejection) and multi-organ diseases (e.g.systemic lupus erythematosis) and inflammatory sequelae of viral orbacterial infections. There is also evidence that INOS inhibitors may beuseful in the prophylaxis or treatment of bacterial infections (e.g.pneumonia), in particular, by reducing bacterial load in an infectedmammal.

Furthermore, there is evidence for overproduction of NO by iNOS inatherosclerosis and following hypoxic or ischaemic insults (with orwithout reperfusion), for example in the brain or in ischaemic heartdisease.

Disorders of gastrointestinal motility include ileus, for examplepost-operative ileus and ileus during sepsis.

By diseases of the central nervous system is meant those for whichoverproduction of NO is implicated, for example migraine, psychosis,anxiety, schizophrenia, sleep disorders, cerebral ischaemia, CNS trauma,epilepsy, multiple sclerosis, AIDS dementia, chronic neurodegenerativedisease (e.g. Lewy Body Dementia, Huntington's disease, Parkinson'sdisease, or Alzheimer's disease) and acute and chronic pain, andconditions in which non-adrenergic non-cholinergic nerve may beimplicated such as priapism, obesity and hyperphagia.

Examples of acute pain include musculoskeletal pain, post operative painand surgical pain. Examples of chronic pain include chronic inflammatorypain (e.g. rheumatoid arthritis and osteoarthritis), neuropathic pain(e.g. post herpetic neuralgia, diabetic neuropathies associated withdiabeties, trigeminal neuralgia, pain associated with functional boweldisorders, e.g. irritable bowel syndrome, non cardiac chest pain andsympathetically maintained pain) and pain associated with cancer andfibromyalgia.

Furthermore, inhibition of NO synthase may be of advantage in preventingthe lymphocyte loss associated with HIV infection, in increasing theradiosensitivity of tumours during radiotherapy and in reducing tumourgrowth, tumour progression, angiogenesis, and metastasis.

Accordingly, the present invention provides a method for the prophylaxisor treatment of a clinical condition in a mammal, such as a human, forwhich an inhibitor of nitric oxide synthase, for example, an iNOSinhibitor is indicated, which comprises administration of atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt, solvate, or physiologically functionalderivative thereof. In particular, the present invention provides amethod for the prophylaxis or treatment of an inflammatory and/or immunedisorder, such as arthritis or asthma. In a preferred aspect the presentinvention provides a method for the prophylaxis or treatment of aclinical condition selected from arthritis, asthma, ileus, and migraine.In a further aspect, the present invention provides a method for theprophylaxis or treatment of a bacterial infection.

In the alternative, there is also provided a compound of formula (I) ora pharmaceutically acceptable salt, solvate, or physiologicallyfunctional derivative thereof for use in medical therapy, particularly,for use in the prophylaxis or treatment of a clinical condition in amammal, such as a human, for which an inhibitor of nitric oxidesynthase, for example an iNOS inhibitor, is indicated. In particular,there is provided a compound of formula (I) or a pharmaceuticallyacceptable salt, solvate, or physiologically functional derivativethereof for the prophylaxis or treatment of an inflammatory and/orimmune disorder, such as arthritis or asthma. In a preferred aspect,there is provided a compound of formula (I) or a pharmaceuticallyacceptable salt, solvate, or physiologically functional derivativethereof for the prophylaxis or treatment of arthritis, asthma, ileus,and migraine. In a further aspect, there is provided a compound offormula (I) or a pharmaceutically acceptable salt, solvate, orphysiologically functional derivative thereof for the prophylaxis ortreatment of a bacterial infection.

The amount of a compound of formula (1), or a pharmaceuticallyacceptable salt, solvate, or physiologically functional derivativethereof which is required to achieve a therapeutic effect will, ofcourse, vary with the particular compound, the route of administration,the subject under treatment, and the particular disorder or diseasebeing treated. The compounds of the invention may be administered orallyor via injection at a dose of from 0.1 to 1500 mg/kg per day, preferably0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 35 g/day and preferably 5 mg to 2 g/day. Tablets or otherforms of presentation provided in discrete units may convenientlycontain an amount of compound of the invention which is effective atsuch dosage or as a multiple of the same, for instance, units containing5 mg to 500 mg, usually around 10 mg to 200 mg.

While it is possible for the compound of formula (I), or apharmaceutically acceptable salt, solvate, or physiologically functionalderivative thereof to be administered alone, it is preferable to presentit as a pharmaceutical formulation.

Accordingly, the present invention further provides a pharmaceuticalformulation comprising a compound of formula (I) or a pharmaceuticallyacceptable salt, solvate, or physiologically functional derivativethereof, and a pharmaceutically acceptable carrier or excipient, andoptionally one or more other therapeutic ingredients.

The present invention also provides the use of a compound of formula(I), or a pharmaceutically acceptable salt, solvate, or physiologicallyfunctional derivative thereof in the manufacture of a medicament for theprophylaxis or treatment of a clinical condition for which an inhibitorof nitric oxide synthase, for example an iNOS inhibitor, is indicated,for example an inflammatory and/or immune disorder, such as arthritis orasthma. In a preferred aspect, there is provided a compound of formula(I), or a pharmaceutically acceptable salt, solvate, or physiologicallyfunctional derivative thereof in the manufacture of a medicament for theprophylaxis or treatment of a clinical condition selected fromarthritis, asthma, ileus, and migraine. In a further aspect, there isprovided a compound of formula (I), or a pharmaceutically acceptablesalt, solvate, or physiologically functional derivative thereof in themanufacture of a medicament for the prophylaxis or treatment of abacterial infection.

Hereinafter, the term “active ingredient” means a compound of formula(I), or a pharmaceutically acceptable salt, solvate, or physiologicallyfunctional derivative thereof.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular), inhalation (including fine particle dusts or mistswhich may be generated by means of various types of metered dosepressurised aerosols, nebulisers or insufflators), rectal and topical(including dermal, buccal, sublingual and intraocular) administrationalthough the most suitable route may depend upon for example thecondition and disorder of the recipient. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. All methods includethe step of bringing the active ingredient into association with thecarrier which constitutes one or more accessory ingredients. In generalthe formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basissuch as gelatin and glycerin or sucrose and acacia.

Preferred unit dosage formulations are those containing an effectivedose, as hereinbefore recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

According to a further aspect of the invention, there is provided aprocess for preparing a compound of formula (I) or a salt, solvate, orphysiologically functional derivative thereof which comprises:

(i) reaction of the compound of formula (II)

or an optical isomer, a salt, or a protected derivative thereof, whereinR¹ is as defined above, with a compound of formula (III)

or a salt thereof, wherein L is a leaving group, most suitably a C₁₋₆alkoxy group, for example ethoxy, or an alkylthio, aralkylthio orarylthio group e.g. a benzylthio, or 1- or 2-naphthylmethylthio group;followed by the following steps in any order:

(ii) optional removal of any protecting groups;

(iii) optional separation of an optical isomer from a mixture of opticalisomers;

(iv) optional conversion of the product to a corresponding salt,solvate, or physiologically functional derivative thereof.

When L is C₁₋₆ alkoxy, the reaction in step (i) above may be effected insolution at alkaline pH, for example pH 8 to 11, suitably at pH 10.5,and at a low temperature, for example −5° C. to 20° C., suitably 0 to 5°C. When L is an alkylthio, aralkylthio, or arylthio group, the reactionmay be effected in an organic solvent e.g. tetrahydrofuran or aC₁₋₄alcohol such as ethanol, at a moderate temperature e.g. 10 to 40°C., suitably at ambient temperature.

Compounds of formula (III) and salts thereof are available commerciallyor may be prepared by methods of organic chemistry well known to theperson skilled in the art, for example, as described by Shearer et al inTetrahedron Letters, 1997, 38, 179-182.

Compounds of formula (II) and salts and protected derivatives thereofmay be prepared from a compound of formula (IV):

or a protected derivative thereof, by coupling with a compound offormula (V)

or a protected derivative thereof, wherein R¹ is as defined above and L¹is a leaving group, for example halo, such as bromo, or an alkyl, arylor aralkyl sulphonate ester, such as toluenesulphonyl.

Protected derivatives of a compound of formula (IV) e.g.N-t-butoxycarbonyl cysteine t-butyl ester may react with compounds offormula (V) under conditions in an appropriate organic solvent (e.g.toluene) in a reaction mediated by a base such as1,8-diazabicyclo[5.4.0]undec-7-ene or a similar agent which would berecognised by one skilled in the art.

Alternatively, compounds of formula (II) and salts and protectedderivatives thereof may be prepared by reduction (for example, using ametal hydride complex) of a compound of formula (VI)

or a protected derivative thereof, wherein R¹ is as defined above.

Compounds of formula (VI) may be prepared by methods analogous to thosedescribed by Yanagisawa et al in J. Med. Chem 30 (11), 1984-91, (1987)and Hassner and Dehaen in J. Org. Chem., 55, 5505-5510, (1990). Thus,reaction of the compound of the formula (IV) or a protected derivativethereof (for example, where the amino group is protected with an acylgroup such as t-butoxycarbonyl and the carboxylic acid group isprotected as an ester, such as a tert-butyl ester) with nitromethane andthe appropriate aldehyde R¹CHO, wherein R¹ is as defined above, in thepresence of piperidine yields a nitro compound of formula (VI) or aprotected derivative thereof.

Compounds of formula (II) or a protected derivative thereof may also beprepared by reaction of a compound of formula (VII)

or a protected derivative thereof wherein R¹ is as defined above, withan aziridine of formula (VIII).

or a protected derivative thereof, for example, wherein the amine isprotected with an acyl group such as t-butoxycarbonyl and the acid isprotected as an ester such as C₁₋₄alkyl ester. The reaction of thecompounds of formulae (VII) and (VIII) may be effected in an inertsolvent, such as chloroform in the presence of a Lewis acid, such asBF₃(OEt)₂.

The compounds of formulae (IV), (V), (VII), and (VIII) and protectedderivatives thereof are commercially available or may be prepared bymethods of organic chemistry well known to the person skilled in theart.

The protecting groups used in the preparation of compounds of formula(I) may be used in a conventional manner, for example, using methodsdescribed in “Protective Groups in Organic Synthesis” by Theodora WGreen, 2nd edition (John Wiley and Sons, 1991) which also describesmethods for the removal of such groups.

In the above reactions, primary amines are suitably protected using acylgroups, such as t-butoxycarbonyl or benzyloxycarbonyl groups which maybe removed under acidic conditions, for example, by treatment withhydrochloric acid or hydrobromic acid, or by hydrogenolysis.

As will be appreciated by the person skilled in the art use of suchprotecting groups may include orthogonal protection of amino groups inthe compounds of formula (II) to facilitate the selective removal of onegroup in the presence of another, thus enabling selectivefunctionalisation of a single amino function. For example, abenzyloxycarbonyl group may be selectively removed by hydrogenolysis. Aperson skilled in the art will also appreciate other orthogonalprotection strategies, available by conventional means as described inTheodora W Green (vide supra).

The enantiomeric compounds of the invention may be obtained (a) byseparation of the components of the corresponding racemic mixture, forexample, by means of a chiral chromatography column, enzymaticresolution methods or preparing and separating suitablediastereoisomers, or (b) by direct synthesis from the appropriate chiralintermediates by the methods described above.

Optional conversion of a compound of formula (I) to a corresponding saltmay conveniently be effected by reaction with the appropriate acid orbase. Optional conversion of a compound of formula (I) to acorresponding solvate or physiologically functional derivative may beeffected by methods known to those skilled in the art.

According to a further aspect, the present invention provides novelintermediates for the preparation of compounds of formula (I), forexample: compounds of formula (II) as defined above, or an opticalisomer, a salt, or a protected derivative thereof; particularly, acompound selected from:

(R,R)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(R,S)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(S,S)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(S,R)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-ethyl-4-thiohexanoate;

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-butoxymethyl-4-thiohexanoate;

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-cyclopropyl-4-thiohexanoate;

(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(R/S)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(S,S)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(S,R)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate;

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-ethyl-4-thiohexanoate;and

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-butoxymethyl-4-thiohexanoate.

In a particular aspect of the invention, the compound of formula (II) isselected from(R,R)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoateand(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate.

Certain protected derivatives of the compounds of formula (VI) are alsouseful as intermediates for the preparation of compounds of formula(II); particularly(R,R/S)-t-butyl-2N-t-butoxycarbonyl-2-amino-5-cyclopropyl-6-nitro-4-thiohexanoate.

Certain protected derivatives of the compounds of formula (I) are alsouseful as intermediates for the preparation of compounds of formula (I);particularly a compound selected from:

(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;

(R,S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;

(S,S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;

(S,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-ethyl-4-thiohexanoate;

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-butoxymethyl-4-thiohexanoate;and

(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-cyclopropyl-4-thiohexanoate;and salts and solvates thereof.

In a particular aspect of the invention, the protected derivative offormula (I) is(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethy)-2,6-diamino-5-methyl-4-thiohexanoateor a salt or solvate thereof.

For a better understanding of the invention, the following Examples aregiven by way of illustration.

SYNTHETIC EXAMPLES Example 1 Synthesis of(R,R)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatedihydrochloride or S-[(R)-2-(1-iminoethylamino)propyl]-L-cysteinedihydrochloride

(a)(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate

To a solution of N-t-butoxycarbonyl cysteine t-butyl ester (7.26 g, 26.2mmol) (Olsen et al., J Med. Chem., 1985, 50 (22), 4332-4336) in drytoluene (100 ml) was added (S)-N-benzyloxycarbonyl 1-aminopropan-2-oltosylate (9.51 g, 26.2 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene(3.90 ml, 26.2 mmol) and the mixture stirred vigorously overnight at 60°C. under nitrogen. The mixture was partitioned between 250 ml each ofethyl acetate and 1N aqueous HCl. A further organic extract was combinedand these extracts were washed with aqueous sodium bicarbonate, waterand brine, then dried and evaporated. Purification by columnchromatography afforded the title compound as a colourless oil whichcrystallised to give a white solid on prolonged standing.

LC/Electrospray Mass Spec., RT 4.93 min, M+H 469 (100%), M+NH₄ ⁺ 486(70%)

¹H NMR (CDCl₃) δ_(H); 1.27 (3H, d, Me), 1.44 and 1.47 (ea 9H, s, CMe₃),2.92 (3H, m, 3-H, 5-H), 3.23 and 3.38 (ea 1H, m, 6-H), 4.39 (1H, brm,2-H), 5.11 (2H, s, CH₂Ph) 5.31 and 5.42 (ea 1H, br, NH), 7.34 (5H, m,Ar-H).

Circular Dichroism spectrum (MeCN)

210 (+0.42) and 233 (−0.11) nm

(b)(R,R)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate

To a solution of(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6-diamino-5-methyl-4-thiohexanoate(5 g) in 120 ml of Ethanol, degassed and blanketed with nitrogen, wasadded 2.5 g of Palladium hydroxide on charcoal (20%, Degussa type E101NE/W, 1:1 with water) followed by ammonium formate (10 g). The solutionwas then heated and refluxed for 1 hour, after which it was cooled andfiltered through hyflo, well-washed with aqueous ethanol. This wasevaporated and the residue passed down a short silica column eluted with90:10:0.5 chloroform: methanol: 880 ammonia, to yield an oil onevaporation which was taken directly to the next stage.

LC/Electrospray Mass Spec., RT 2.40 minutes, M+H 335 (100%), 279 (60%)

(c)(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatehydrochloride.

A single portion of S-(1-naphthylmethyl)thioacetimidate hydrochloride(3.10 g, 12.3 mmol) was added to a solution of(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-2,6-diamino-5-methyl-4-thiohexanoate2.75 g in 50 ml of ethanol under nitrogen and the solution stirred for14 h at room temperature. The solvent was evaporated and the residuepartitioned between 50 ml each of ether and water, followed by 2 etherwashes; back aqueous extracts were combined and evaporated to give acrude white amorphous paste which was used directly in the next stage.

Thermospray Mass spectrum M+H 376 (100%), 276 (12%).

¹H NMR (D₂O) δ_(H); 1.28 (3H, d, Me), 1.39 and 1.42 (ea 9H, s, CMe₃),2.21 (3H, CH₃) 3.01 (3H, m, 3-H, 5-H), 3.37 (2H, m, 6-H), 4.18 (1H, t,2-H).

(d) (R,R)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatedihydrochloride;

or S-[(R)-2-(1-iminoethylamino)propyl]-L-cysteine dihydrochloride

(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatehydrochloride was dissolved by the addition of 20 ml of 4N hydrogenchloride in dioxane and the soon-formed suspension vigorously stirredovernight under nitrogen. 50 ml of ether was added and the liquid wasdecanted form the sticky gum. Trituration with ether ultimately affordedthe title compound as an amorphous hygroscopic white solid.

Electrospray Mass spectrum M+H 220 (100%),

^(H) NMR (D₂O) δ_(H); 1.37 (3H, d, Me), 2.25 (3H, s, CH₃) 3.23 (3H, m,3-H, 5-H), 3.47 (2H, m, 6-H), 4.24 (1H, t, 2-H) (note that there is somedoubling of signals due to rotameric forms).

¹³C NMR (DMSO-d₆) δ_(C); 18.90 (q, Me), 19.30 (q, Me), 30.13 (t, 3-C),38.83 (d, 5-C), 47.43 (t, 6-C) 52.20 (d, 2-C), 165.0 (s, N=C-N), 170.0(s, CO₂H).

Circular Dichroism spectrum (H₂O)

199 (+1.38) and 223 (−0.77) nm

Example 2 Synthesis of(R,S)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatedihydrochloride or S-[(S)-2-(1-iminoethylamino)propyl]-L-cysteinedihydrochloride

The procedures and methods were identical to those used in Example 1,except that in the alkylation step, protected L-Cysteine was reactedwith (R)-N-benzyloxycarbonyl 1-aminopropan-2-ol tosylate instead of the(S)-enantiomer of that example.

Electrospray Mass spectrum M+H 220 (100%),

¹H NMR (D₂O) δ_(H); 1.40 (3H, d, Me), 2.25 (3H, s, CH₃) 3.30 (3H, m,3-H), 5H), 3.45 (2H, m, 6-H), 4.25 (1H, t, 2-H) (n.b. some doubling ofsignals due to rotameric forms).

Example 3 Synthesis of(S,S)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatedihydrochloride or S-[(S)-2-(1-iminoethylamino)propyl]-D-cysteinedihydrochloride

The procedures and methods were identical to those used in Example 1,except that in the alkylation step, the enantiomeric protectedD-Cysteine was reacted with (R)-N-benzyloxycarbonyl 1-aminopropan-2-oltosylate.

Product spectra identical to those of compound in Example 1, except:

Circular Dichroism spectrum (H₂O)

199 (−1.05) and 224 (+0.66) nm

Example 4 Synthesis of(S,R)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoatedihydrochloride or S-[(R)-2-(1-iminoethylamino)propyl]-D-cysteinedihydrochloride

The procedures and methods were identical to those used in Example 1,except that in the alkylation step, the enantiomeric protectedD-Cysteine was reacted with (S)-N-benzyloxycarbonyl 1-aminopropan-2-oltosylate.

Electrospray Mass spectrum M+H 220 (100%),

¹H NMR (D₂O) δ_(H); 1.40 (3H, d, Me), 2.25 (3H, s, CH₃) 3.30 (3H, m,3-H, 5H), 3.45 (2H, m, 6-H), 4.25 (1H, t, 2-H)

Example 5 Synthesis of(R,R/S)-6N-(1-iminoethyl)-2,6-diamino-5-ethyl-4-thiohexanoatedihydrochloride or S-[(R/S)-2-(1-iminoethylamino)butyl]-L-cysteinedihydrochloride

The procedures and methods were identical to those used in Example 1,except that in the alkylation step, protected L-Cysteine was reactedwith (R/S)-N-benzyloxycarbonyl 1-aminobutan-2-ol tosylate instead of the1-aminopropan-2-ol derivative of that example, to furnish a product thatwas substantially an epimeric mixture of the title compound.

Electrospray Mass spectrum M+H 234 (100%), Example 6 Synthesis of(R,R/S)-6N-(1-iminoethyl)-2,6-diamino-5-hydroxymethyl-4-thiohexanoatedihydrochloride orS-[(R/S)-2-(1-iminoethylamino,3-hydroxy)propyl]-L-cysteinedihydrochloride

The procedures and methods were identical to those used in Example 1,except that in the alkylation step, protected L-Cysteine was reactedwith (R/S)-N-benzyloxycarbonyl-3-t-butoxy-1-aminopropan-2-ol tosylateinstead of the 1-aminopropan-2-ol derivative of that example. Thisfurnished a product that was substantially an epimeric mixture of thetitle compound with concomitant loss of the tert butyl ether in thedeprotection stage.

Electrospray Mass spectrum M+H 236 (100%),

Example 7 Synthesis of S-[(R/S)-2-(1-iminoethylamino,2-cyclopropyl)ethyl]-L-cysteine dihydrochloride orS-[(R/S)-2-(1-iminoethylamino, 2-cyclopropyl)ethyl]-L-cysteinedihydrochloride

a)(R,R/S)-t-butyl-2N-t-butoxycarbonyl-2-amino-5-cyclopropyl-6-nitro-4-thiohexanoate

Protected L-Cysteine was reacted with nitromethane, piperidine andcyclopropanecarboxaldehyde using the conditions described by Hassner andDehaen in J. Org. Chem., 55, 5505-5510, (1990), affording the titlecompound as a colourless oil.

¹H NMR (CDCl₃) δ_(H); 0.40 and 0.68 (ea 2H, m, cyclopropyl-H), 0.89 (1H,m, cyclopropyl-H), 1.46 and 1.48 (ea 9H, s, CMe₃), 2.81, 3.00 and 3.10(ea 1H, m, 3-H, 5-H), 4.41 (1H, brm, 2-H), 4.58 (2H, m, 6-H), 5.34 (1H,br, NH).

b)(R,R/S)-t-butyl-2N-t-butoxycarbonyl-2,6-diamino-5-cyclopropyl-4-thiohexanoate

The nitro groups of the intermediate from step a) was reduced using amixture Nickel Chloride/Sodium Borohydride in methanol according to themethod described by Nagarajan and Ganem, J. Org. Chem., 51, 4856-4861,(1990). An oil was recovered after silica gel solid phase extraction,which was used directly in the next stage.

c) Synthesis of(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-cyclopropyl-4-thiohexanoate

The product from the reduction step b) (47 mg, 0.13 mmol) above wasreacted with S-(1-naphthylmethyl)thioacetimidate hydrochloride (72 mg)as described in step c) of Example 1 to yield a crude white foam.

¹H NMR (D₂O) δ_(H); 0.05 and 0.34 (ea 2H, m, cyclopropyl-H), 0.59 (1H,m, cyclopropyl-H), 1.09 and 1.12 (ea 9H, s, CMe₃), 1.92 (3H, s Me), 2.05(1H, m, 5-H), 2.70 (2H, m, 3-H), 3.25, (2H, m, 6-H) 3.89 (1H, m, 2-H).

Electrospray Mass spectrum M+H 402 (50%),

d) Synthesis of S-[(R,S)-2-(1-iminoethylamino,2-cyclopropyl)ethyl]-L-cysteine dihydrochloride orS-[(R/S)-2-(1-iminoethylamino, 2-cyclopropyl)ethyl]-L-cysteinedihydrochloride

The deprotection of(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-cyclopropyl-4-thiohexanoatewas carried out using 4N HCl in dioxane. The product, a mixture ofepimers at C-5, was isolated as a hygroscopic glassy solid after a C-18solid phase extraction eluted with water.

¹H NMR (D₂O) δ_(H); 0.29 and 0.58 (ea 2H, m, cyclopropyl-H), 0.82 (1H,m, cyclopropyl-H), 2.14 (3H, s Me), 2.34 (1H, m, 5-H), 3.12 (2H, m,3-H), 3.48, (2H, m, 6-H) 3.98 (1H, m, 2-H).

Electrospray Mass spectrum M+H 246 (100%),

Biological Activity

1. Inhibition of Isolated Human iNOS

The inhibition of purified human iNOS may be determined using apreparation of human iNOS as described in the chapter “Expression ifHuman Nitric Oxide Synthase Isozymes” by Charles et al in Methods inEnzymology, 1996 Volume 268, 449-60. Activity may be monitored usingquantitative absorption changes of haemoglobin as described by R. G.Knowles and J. Dawson in “A Microtitreplate Assay of Human NOS Isoforms”in Methods in Molecular Biology, 1998, Volume 100, 237-242, Nitric OxideProtocols, Ed M. A. Titheradge, Humana Press, Totowa N.J.

Compound IC50 (μM) Example 1 2.0 Example 2 39.0 Example 3 6.6 Example 448.0 Example 7 6.2

2. Inhibition of eNOS and iNOS in Rat Aortic Rings

The inhibition of eNOS and iNOS in situ in rat aortic rings was assessedby measuring the increases in ring tension caused by NO synthaseinhibition. For studies of basal tone (reflecting eNOS), rings ofthoracic aorta with intact endothelium were prepared as describedpreviously (Rees et al. (1989) Br. J. Pharmol. 96, 418-24) andcumulative concentration curves obtained for the inhibitors in thepresence of a threshold concentration of phenylephrine (ED₁₀≈10 nM). Forstudies of induced smooth muscle tone (reflecting iNOS),endothelium-denuded rings were exposed to LPS (0.1 μg/ml from S.typhosa)in the presence of phenylephrine at approximately ED₉₀ for 6h asdescribed previously (Rees et al. (1990) Biochem. Biophys. Res. Commun.173, 541-547). During this time a progressive loss of tone occurredbecause of iNOS induction. Cumulative concentration curves were thenobtained for the inhibitors.

The results are given in the following table:

iNOS eNOS selectivity IC₅₀ (μM) IC₅₀ (μM) iNOS vs eNOS Example 10.26 >20 >75

3. Inhibition of nNOS in Rat Cortical Slices

The effects of compounds on nNOS in rat brain slices was determined asdescribed in Furfine et al(1994) J. Biol. Chem. 269, 26677-26683 andLizasoain et al (1995) J. Neurochem. 64, 636-642.

KCl (54 mM)—stimulated NO synthesis was measured by the conversion of14C-arginine to 14C-citrulline over a 2h period at 37° C. inMcllwain—chopped (0.2 mm×0.2mm) rat cerebral cortex slices, following a1 h preincubation period in the absence of compound or high KCl.

The compound of Example 1 was determined to have an IC₅₀ of >80 μM,suggesting approximately >300-fold selectivity for iNOS versus nNOS.

4. Method for Determining the Oral Bioavailability of iNOS InhibitorCompounds

Animal Work

Rats (3 animals per time point) were dosed intravenously (10 mg/kg) andorally (50 mg/kg) with test compound in an aqueous solution. Bloodsamples were taken at time intervals after administration and plasmaprepared by centrifugation. Samples were stored at −20° C. untilanalysis.

Analysis of Compounds in Plasma

Plasma (50 μl) was de-proteinated and compound derivatised with aquaternary ammonium reagent. Samples were then injected onto an HPLCsystem and compound concentration determined using mass spectrometricdetection.

Pharmacokinetic Analysis

The plasma concentrations obtained by the above method were entered intoa pharmacokinetic software package (PKCAL v 1.2s) and the data werefitted using a non-compartmental method. The oral bioavailability of thecompounds was determined by comparing the Area Under the Curve (AUC)values calculated by the software for the oral profile with the AUC forthe intravenous profile. The half-lives were obtained by fitting theterminal phase time points of the intravenous profile.

The compound of Example 1 was found to have an oral bioavailabilityof >90% and a half-life of 2-4 hours.

What is claimed is:
 1. A compound of formula (I);

or a salt, solvate, or physiologically functional derivative thereof;wherein R¹ is selected from the group consisting of C₁₋₄ alkyl, C₃₋₄cycloalkyl, C₁₋₄ hydroxyalkyl, and C₁₋₄ haloalkyl.
 2. A method for theprophylaxis or treatment of a clinical condition in a mammal for whichan inhibitor of nitric oxide synthase is indicated, which comprisesadministration of a therapeutically effective amount of a compound offormula (I) as defined in claim 1, or a pharmaceutically acceptablesalt, solvate, or physiologically functional derivative thereof.
 3. Amethod according to claim 2 wherein the clinical condition is selectedfrom the group consisting of arthritis, asthma, ileus, and migraine. 4.A pharmaceutical formulation comprising a compound of formula (I) asdefined in claim 1 or a pharmaceutically acceptable salt, solvate, orphysiologically functional derivative thereof, and a pharmaceuticallyacceptable carrier or excipient, and optionally one or more othertherapeutic ingredients.
 5. A process for preparing a compound offormula (I);

or a salt, solvate, or physiologically functional derivative thereof;wherein R¹ is selected from the group consisting of C₁₋₄ alkyl, C₃₋₄cycloalkyl, C₁₋₄ hydroxyalkyl, and C₁₋₄ haloalkyl; said processcomprising the steps of: (i) reacting the compound of formula (II)

or an optical isomer, a salt, or a protected derivative thereof with acompound of formula (III)

or a salt thereof, wherein L is a leaving group; followed by thefollowing steps in any order: (ii) optional removing any protectinggroups; (iii) optional separating an optical isomer from a mixture ofoptical isomers; and (iv) optional converting the product to acorresponding salt, solvate, or physiologically functional derivativethereof.
 6. A compound of formula (II)

or an optical isomer, a salt, or a protected derivative thereof, whereinR¹ is selected from the group consisting of C₁₋₄ alkyl, C₃₋₄ cycloalkyl,C₁₋₄ hydroxyalkyl, and C₁₋₄ haloalkyl.
 7. A protected derivative of acompound of formula (I) according to claim 1 which is selected from:(R,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;(R,S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;(S,S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;(S,R)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate;(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-ethyl-4-thiohexanoate;(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-butoxymethyl-4-thiohexanoate;and(R,R/S)-t-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamino-5-cyclopropyl-4-thiohexanoate.8. A compound of selected from the group consisting of:S-((R)-2-(1-iminoethylamino)propyl)-L-cysteine;S-((S)-2-(1-iminoethylamino)propyl)-L-cysteine;S-((R/S)-2-(1-iminoethylamino)propyl)-L-cysteine;S-((R)-2-(1-iminoethylamino)propyl)-D-cysteine;S-((S)-2-(1-iminoethylamino)propyl)-D-cysteine;S-((R/S)-2-(1-iminoethylamino)propyl)-D-cysteine;S-((R/S)-2-(1-iminoethylamino)butyl)-L-cysteine;S-((R/S)-2-(1-iminoethylamino,2-cyclopropyl)ethyl)-L-cysteine; andS-((R/S)-2-(1-iminoethylamino,3-hydroxy)propyl)-L-cysteine, or apharmaceutically acceptable salt, solvate, or physiologically functionalderivative thereof.
 9. S-((R)-2-(1-iminoethylamino)-propyl)-L-cysteineor a pharmaceutically acceptable salt, solvate or physiologicallyfunctional derivative thereof.